Background Haptoglobin (HPT) is a plasma protein that binds free hemoglobin with high affinity and thereby inhibits its oxidative activity. HPT exists in two allelic forms in the human population, which produces three known phenotypes. These depend on the HPT alpha chains that are expressed (α1, α2 or both), whereas the β chain is always present. In a previous proteomic study by our group, the three different HPT chains were identified as altered in the serum of patients suffering osteoarthritis (OA).
Objectives The aim of this study is to verify the usefulness of haptoglobin chains as serum biomarkers for osteoarthritis disease using two independent methods.
Methods The abundance of the three different HPT chains was evaluated by Western blotting on 30 serum samples, 15 OA and 15 controls. The samples were obtained from anonymous donors in the Complejo Hospitalario Universitario A Coruña, Spain. The Western blot analysis was performed with standard 15% polyacrylamide SDS-PAGE gels and monoclonal antibodies against the β, α-1 and α -2 chains of haptoglobin. The relative abundance of HPT chains was calculated by obtaining the ratio of normalized densitometric values between OA and normal samples. To verify the alteration of HPT α-2 chain in OA sera versus controls by an orthogonal method, additional OA and control samples were previously depleted with a chemical sequential depletion method developed in our lab. Proteins in these samples were digested and analyzed by multiple reaction monitoring technology (MRM) using a nanoLC coupled to a 5500 QTRAP mass spectrometer. This experiment was developed by triplicate per sample, and the mean areas obtained were compared in the OA samples versus control. A standard of beta-galactosidase was used as internal standard.
Results Using Western blot analysis we detected the increase of the HPT β chain in OA sera, and relatively quantified this increase as 2.22-fold by densitometric analysis of the blots (p=0.017). We also found that the HPT α-1 chain was 1.4-fold increased in OA sera (p=0.012), whereas the HPT α-2 chain was 0.7 decreased in OA sera versus control, although without statistical significance. Using the MRM technique, we relatively quantified the decrease of the HPT α-2 chain with a normalized area 0.4-fold decreased in OA vs control (p=0.009). With these data, we verified the OA-dependent increase of HPT α-1 and β by Western blot analysis, and the decrease of α-2 chain was observed by two different methods, MRM and Western blot.
Conclusions We have been able to verify for the first time the OA- dependent alteration of the haptoglobin chains. These data suggest that these chains might be potential OA biomarkers in sera. A further analysis on larger sample cohorts will be carried out to validate their sensitivity and specificity.
Disclosure of Interest : None declared